Project Summary/Abstract Mu opioid receptors play a fundamental role in mediating the actions of morphine and most clinical analgesics, as well as drugs of abuse, such as heroin. The single-copy mu opioid receptor gene (OPRM1) creates an array of splicing variants by undergoing extensive alternative pre-mRNA splicing (AS), that is conserved from rodents to humans. These splice variants are categorized into three types based on receptor structure: 1) Full- length carboxyl (C-) terminal variants with 7-transmembrane (TM) domains; 2) Truncated variants containing 6- TM domains; and 3) Truncated variants containing single TM. Increasing evidence suggests that the OPRM1 AS variants are pharmacologically important. Several C-terminal variants display marked differences in region- specific expression, mu agonist-induced G protein coupling, phosphorylation, internalization and post- endocytic sorting. MOR-1D is responsible for morphine-induced itch. Dysregulation of several variant mRNA expressions has been observed in a number of cell and animal models, as well as human diseases. Evidence from our three knockout mouse models suggest that individual C-terminal sequences generated through alternative 3' splicing play distinct roles in various morphine actions, including tolerance, physical dependence and reward. Additionally, intracerebroventricular (i.c.v.) administration of an antisense vivo-morpholino antisense oligo, which blocks 3' splicing from exon 3 to exon 7, significantly attenuates morphine tolerance in mice. Together, these studies not only strongly support our hypothesis that OPRM1 alternative splicing contributes to the regulation of complex opioid actions in animals and humans, but also provide a compelling rationale and scientific premise for further study of the molecular mechanisms controlling OPRM1 alternative splicing and assessing the impact of modulating OPRM1 alternative splicing using antisense vivo-morpholino oligos on morphine actions, as proposed in this application. The primary goal of this application is to further investigate mechanisms and functions of OPRM1 gene alternative splicing by using a variety of in vitro and in vivo approaches. The specific aims include: 1) Decoding molecular mechanisms underlying OPRM1 3' splicing by identifying cis-acting elements and trans-acting factors that regulate the 3' splicing; 2) Investigating the role of the OPRM1 3' splicing in mu opioid actions in both Be(2)C cells and mice using an antisense vivo- morpholino oligo approach. The proposed studies promise to generate significant insights into the mechanism and function of OPRM1 3' splicing, and may have the potential for developing new therapeutics for controlling pain and alleviating the detrimental side-effects of mu opioids.